1. The Field of Invention
This invention relates to an apparatus and method for conducting fluorometric measurements and more particularly to homogeneous fluorescent immunoassays. Finally, it relates to the determination of the presence and concentrations of a plurality of fluorescing bodies (and indirectly non-fluorescing bodies) simultaneously and homogeneously.
2. The Prior Art
Fluorescence technology has become widespread in the fields of clinical laboratory testing, research and medical diagnostic testing areas. It is widely regarded as a technique for making very sensitive and specific test determinations, competing effectively in many areas with radioimmunoassays and enzyme immunoassays.
Fluorescence is the physical phenomenon occurring when a molecule or atom is bombarded with light of given wavelengths; namely the conversion of that light to an emission of light of a different wavelength. In macroscopic terms, the conversion is instantaneous, but in real terms the finite time differences between the absorption of the light by the molecule and the time interval during which the emitted light is given off is a measure of the characteristics of the bodies being measured.
The process of fluorescence starts with the absorption of light photons by atoms or molecules. The frequency of light absorption varies with the atom or molecule involved.
Fluroescent molecules in any specific environment have two characteristic spectra. The first, the so-called excitation spectrum, is represented by a series of wavelengths of light which are absorbed by the molecule with differing efficiences. That is, out of a possible number of existing wavelengths which may be absorbed by the molecule to cause fluorescence, usually one of these will be absorbed at a greater level. Most atoms or molecules that absorb light convert this light energy into heat, but a few emit light or "fluoresce" at a lower light frequency. Photon absorption occurs rapidly in about 10.sup.-15 seconds. If the light excitation is abruptly interrupted, as with a very short pulse of light, photon light emission, in the second spectrum will decay rapidly with a time constant that depends on the atom or molecule involved. The range of decay times is usually between 10.sup.-10 to 10.sup.-6 seconds (0.1 to 1000 nanoseconds). The intensity of the emission spectrum is directly proportional to the intensity of the exciting light.
It happens also that the intensity of the emitted light is also directly proportional to the concentration of the fluorescent molecules in the sample. It thus can be seen that a very sensitive technique for measuring the concentration of a fluorescent body can be evolved by controlling the intensity of the exciting light and other physical constants of the measuring system.
The analytical value of fluorescence decay time measurement arises from the fact that each atom or molecule has its own distinctive rate of decay. Each atom or molecule is excited at a different frequency and emits light only at a particular emission wavelength. Problems arise, however, when substances under test have overlapping emission wavelengths. In these instances, decay time measurement often becomes the only means of discrimination between differing fluorescing bodies.
An example of such overlapping emission wavelengths occurs in clinical tests of human blood serum. A variety of substances in blood serum fluoresce at the same wavelengths as the fluorochromes used in fluoroimmunoassay. Fortunately, these blood serum components have very short decay times (of the order of 1-10 nanoseconds), and using a decay time measurement, the substance of interest, such as a fluorochrome in fluoroimmunoassay, may be separately determined by these differences in decay times and by measuring the emission at wavelengths substantially different from the emission wavelengths of the background. This has not been an altogether satisfactory technique, however.
More recently the art has sought to take advantage of the decay characteristics of the emission spectra of a fluorescing body in an attempt to discriminate background fluorescence from the fluorescing body under study. For example, in U.S. Pat. No. 4,058,732 issued to Weider on Nov. 15, 1977, the inventor describes the excitation of target molecules (the substances to be measured) tagged with a fluorescent tag having a relatively long fluorescent decay lifetime compared to the decay lifetime of ambient or background substances. A fluorescent detection system is programmed to read the resulting fluorescence only after the fluorescence of the background has substantially decayed. Thus, the only fluorescence detected is that of the tagged substance, the ambient fluorescence having been allowed to disappear. The technique requires in addition that the excess fluorescent tag be separated from the tagged substance to be measured. More about this type of technique will be described below. The decay time measurements are taken over a short time interval in the nanosecond (10.sup.-9 seconds) range usually 2-150 nanoseconds. Typically, ambient decay times are about 2-10 nanoseconds with specific tagged fluorescent material being in the range of 15 to 100 nanoseconds.
U.S. Pat. No. 4,006,360 to Mueller issued Feb. 1, 1977 also utilizes the longer decay lifetimes of bound fluorescing materials. This technique similarly utilizes a time-gating to discriminate between emissions from two populations of fluorescent dye molecules which exhibit different excited state lifetimes. In addition, the technique includes measuring the fluorescence over a predetermined time interval.
Similarly, "Biochemical Applications of a Synchronously Pumped Krypton Ion Dye Laser Flouorescence System," by Richardson et. al., Analytical Biochemistry 97, 17-23 (1979), describes other populations of fluorescent dye molecules which exhibit different excited state lifetimes depending on whether they are free, or attached to antibodies or antigen/antibody complexes.
U.S. Pat. No. 4,259,574 issued Mar. 31, 1981 to Carr & Froot relates basically to the determination of impurities or amalgams in semiconductor materials as measured by changes in the decay lifetime of the contaminated semiconductors versus a record of known fluorescent decay rates of the pure, unaltered molecular semiconductor.
There are a wide variety of prior art techniques which can be used to conduct immunoassays irrespective of the particular tag or label used. Typically, an immunoassay may involve, for example, competitive binding in which the antigen (drug, hormone, virus, or prokaryotic, eukaryotic or somatic cells, for example) to be determined, and its corresponding antibody are combined with the identical antigen labelled appropriately. Both the labelled and nonlabelled antigens compete for antibody binding sites. The amount of labelled antigen which binds to the antibody is dependent upon, and therefore a measurement of, the concentration of nonlabelled antigen. The determination requires separation of the reacted labelled antigen from the unreacted labelled antigen. This is thus termed a heterogeneous assay.
In another method, the so-called sandwich method, the antibody is bound to a suitable solid support, such as the wall of a plastic test tube, small glass or plastic beads, or SEPHADEX particles (to facilitate later separation). This solid, antibody-coated material is then exposed to a solution of the unknown antigen, permitting antigen/antibody reaction to take place at the solid phase surface. The surface is washed and exposed to a solution of labelled antibody specific for the unknown antigen, permitting its binding with the exposed antigen. Excess unreacted labelled antibody is separated from the bound material, leaving the remaining fluorescence as a measurement of the target antigen in the biological sample.
There are several other formats for immunoassays some of which require separation of the unreacted tagged material from the reached tagged complexes.